Mineral substrate quality determines the initial soil microbial development in front of the Nordenskiöldbreen, Svalbard

Abstract Substrate geochemistry is an important factor influencing early microbial development after glacial retreat on nutrient-poor geological substrates in the High Arctic. It is often difficult to separate substrate influence from climate because study locations are distant. Our study in the retreating Nordenskiöldbreen (Svalbard) is one of the few to investigate biogeochemical and microbial succession in two adjacent forefields, which share the same climatic conditions but differ in their underlying geology. The northern silicate forefield evolved in a classical chronosequence, where most geochemical and microbial parameters increased gradually with time. In contrast, the southern carbonate forefield exhibited high levels of nutrients and microbial biomass at the youngest sites, followed by a significant decline and then a gradual increase, which caused a rearrangement in the species and functional composition of the bacterial and fungal communities. This shuffling in the early stages of succession suggests that high nutrient availability in the bedrock could have accelerated early soil succession after deglaciation and thereby promoted more rapid stabilization of the soil and production of higher quality organic matter. Most chemical parameters and bacterial taxa converged with time, while fungi showed no clear pattern.


Introduction
Retr eating ice fr onts, induced by global climate change , ha ve incr easingl y exposed landsca pes in polar and alpine regions, leading to microbial and plant colonization and soil formation (Hodkinson et al. 2003, Bajerski and Wagner 2013, Bradley et al. 2014 ).Microbes are the pioneer colonizers of these deglaciated barren surfaces (Sc hütte et al. 2010, Br adley et al. 2014 ).Their distributions are determined by nutrient availability, and at the same time, they drive nutrient and mineral cycling during the initial phases of soil stabilization and plant establishment (Hodkinson et al. 2003, Tsc herk o et al. 2003, Borin et al. 2010 ).As suc h, c hr onosequence a ppr oac hes hav e pr ovided a useful tool to investigate the environmental factors that may affect the micr obial comm unity during ecosystem de v elopment (Bernasconi et al. 2011, Zumsteg et al. 2012, Knelman et al. 2014, Garrido-Benavent et al. 2020 ).
Plant colonization and plant-micr obe inter actions hav e been identified as the predominant factors that shape microbial community composition and nutrient accumulation along alpine and low latitude c hr onosequences (Tsc herk o et al. 2003, Zumsteg et al. 2012, Brown and Jumpponen 2014 ).In comparison, polar for efields mainl y comprise cryptogr ams, lic hens and bryophytes (Garrido-Benavent et al. 2020 ), and may remain mostly devoid of plants e v en a century after glacier r etr eat (Br adley et al. 2016 ).Even without vascular plants, shifts of microbial community occur in polar regions over short time intervals (Nemergut et al. 2007, Garrido-Benavent et al. 2020, Vimercati et al. 2022 ), though the possible abiotic driving factors and their complex interaction with carbon sources are still being debated (Tsc herk o et al. 2003, Knelman et al. 2014 ).Together with plants, soil age has also been suggested to be a k e y factor affecting the microbial life and eda phic pr operties (Noll andWellinger 2008 , Br adley et al. 2014 ), though environmental disturbances may ov er print the effect of time (Kim et al. 2017 ).The selective pressure of the environmental factors (local geographic and climatic conditions) and the physico-chemical properties of the substrate (e.g.soil texture and porosity, nutrient status, pH, and mineralogical properties) (Lazzaro et al. 2010 ) together with accessibility to pioneer sites (Jumpponen et al. 1999 ) affect substr ate av ailability and thereby microbial distribution in the glacier forefield (Zhou et al. 2002 ).In general, the initial phases of succession select for microbes that are able to fix carbon and nitrogen or otherwise gain the nutrients from weathering (Wojcik et al. 2019 ), together with het-er otr ophs thriving on available organic nutrient sources (Bardgett et al. 2007, Schulz et al. 2013 ).The proportion and decomposition of old, microbial necromass gradually increases, leading to the recycling of accumulated nutrients (Bradley et al. 2014 ).Recently, among all the possible dri vers, low n utrient availability was suggested to be the main constraining factor for microbial comm unity de v elopment, whic h may riv al other eda phic pr operties and harsh climatic conditions of glacier forefields (Knelman et al. 2014, Schmidt et al. 2016, Dar c y et al. 2018 ).That is, a lack of available nutrients could impose stronger constraints on micr obial comm unity de v elopment in earl y succession than the absence of carbon substrates (i.e.energy source) (Castle et al. 2017 ).The main factors that determine nutrient availability of newly exposed oligotrophic polar soils remain largely unexplored but are pr esumabl y r elated to soil bedr oc k type (Barton et al. 2007, Lazzaro et al. 2009, Tytgat et al. 2016, Wojcik et al. 2019 ) and/or soil organic matter sources (Fierer et al. 2010 ).
Bedr oc k type not only determines physical properties of the landsca pe, suc h as textur e, por osity (Lazzar o et al. 2009 ), and the rate of nutrient release due to weathering (Tytgat et al. 2016 ), but also provides minerals that are known to select for specialized bacteria (Carson et al. 2009, Meola et al. 2014 ).In this way, the bedr oc k type contr ols micr obial habitat and r elease of nutrients, and ther eby significantl y influences micr obial activity in the initial, plant-free phases of soil development (Carson et al. 2009 ).In particular, phosphorus availability has been suggested to limit succession in cold and arid climates with lo w w eathering rates (Dar c y et al. 2018 ).
T he interpla y of life strategies emplo y ed b y microbial colonizers is depicted by the distinct successional trajectories that have been described for bacteria and fungi (Rime et al. 2016b, Jiang et al. 2018, Garrido-Benavent et al. 2020, Vimercati et al. 2022 ).This may be caused by ecological differ ences, suc h as substrate utilization or stress tolerance of fungi and bacteria (Brunner et al. 2011, Hannula et al. 2017 ), which are reflected in the highly specific r equir ements (Rime et al. 2016b ) and mor e r andom distribution (Brown and Jumpponen 2014 ) of fungi in early succession compared to bacteria.Accordingly, taxonomically diverse bacterial comm unities e volving on unr elated glacier for efields with differ ent geoc hemistries hav e been shown to conv er ge with incr easing age of succession (Castle et al. 2016 ), while fungi do not exhibit consistent patterns (Brown and Jumpponen 2014 ).
The present study combines fungal and bacterial successional pathways together with substrate geochemistry to improve our understanding of glacial forefield colonization processes and the implications for future glacier retreat.While most geographical and soil forming conditions of the two forefield chronosequences in front of Nordenskiöldbreen (Svalbar d, Norw ay) are similar, differences in mineral substrate composition offer the possibility to test the effects of contrasting bedrock type and glacial landscape.We, ther efor e, sampled along the contrasting 80-year successional High Arctic glacial forefield gradients, with the assumption that the controls imposed by physico-chemical properties of the miner al substr ate on bacterial and fungal successional tr ajectories are mitigated with soil age.

Our hypotheses are:
(i) There is a significant influence of mineral substrate and different quality of organic matter on the microbial assembly in the first stages of the soil development.(ii) The influence of the mineral substrate and organic matter on the microbial assembly is mitigated with soil age and the patterns of response are different for bacteria and fungi.

Study sites and sample collection
Nordenskiöldbr een (centr al Spitsber gen, 78.67  (Ew erto wski et al. 2016, Allaart et al. 2018 ).The middle part of glacier tongue is a tidewater terminus and both lateral margins are terrestrial.Nordenskiöldbreen has been retreating since the Little Ice Age (LIA) maximum at the end of the 19th century (Allaart et al. 2018 ).Between 1896 and 2013, the northern terrestrial terminated margin r etr eated about 1.4 km, while the southern terrestrial terminated mar gin r etr eated about 3.5 km (Ew erto wski et al. 2016 ).The ne wl y exposed glacier forefields rises from 2 to 35 m a.s .l. T he southern and northern forefields differ in bedr oc k geology and glacial landscape .T he northern silicate forefield is made up of metamorphic r oc ks (primaril y mica sc hist, calc-pelitic sc hists, and marble).
The relief is dominated by ice-moulded bedrock, which is covered by a thin and discontinuous glacigenic and glaciofluvial deposits comprising monotonous clastic material (mainly gneiss and other metamorphites) (Dallmann et al. 2004, Allaart et al. 2018 ).The southern carbonate-silicate forefield is flat and made up of sedimentary carbonates and fewer mica schists .T he bedrock is almost completely covered by thick glacigenic deposits of variable and exotic clastic compositions (granitoids, mafic magmatite, and div erse metamor phites; Dallmann et al. 2004 ).
The annual mean surface ground temperature of the Billefjorden tundr a, r eferr ed to as a r efer ence site, is −5.2 • C, r anging between −19.5 • C for April and 10.8 • C for July, and the annual precipitation is typically less than 200 mm (Førland et al. 2011, Láska et al. 2012 ).Vegetation cover at both forefields is initially absent or sparse (0%-20% vegetation cover, data not shown), consisting primarily of soil crusts and/or single pioneer plants (e.g. S axifraga oppositifolia , Br aya purpur ascens , Salix polaris , and Dryas octopetala) on wetter and wind shielded spots.Tundra reference sites are cover ed pr edominantl y by Carex spp.gr asses and woody shrubs ( S. polaris and D. octopetala).
Soils were collected in July 2015 and 2016 from both northern silicate (N1, N2, N3, and NR sites) and southern carbonate-silicate (S1, S2, S3, and SR sites) forefields with a chronosequence appr oac h (Cr oc ker and Major 1955, Nemer gut et al. 2007, Bernasconi et al. 2011 ).A total of four zones with incr easing a ge wer e sampled from the glacier front (I.0-25, II.26-54, and III.55-79 years old; Fig. 1 ) to the tundra 'reference site' behind the glacier front mor aine fr om the end of the LIA (IV. 10 000 y ears).Zones w ere determined in ArcMap 10.6.1 (ESRI 2018 ) based on aerial photogr a phs fr om the Norwegian Polar Institute fr om the years 1936, 1961, 1990, and 2009(Norsk Polar Institute 2018 ).Proglacial systems are characterized by high spatial variability and redeposition (Hodkinson et al. 2009, Bernasconi et al. 2011 ), ther efor e, the sampling sites were chosen carefully with an effort to find stable surfaces .T he sites were first randomly chosen in ArcMap, checked for potential disturbances by glacier streams on the old aerial photos and then again examined by a geologist in the field with the special focus on the visible glacier mov ement.Miner al surface soil samples (top 5 cm) were collected from unvegetated areas or after the r emov alof soil crusts when pr esent.At e v ery site, thr ee independent soil samples were pooled from four subsamples, each one meter apart (all together 66 mixed samples, 33 for each forefield; Fig. 1 ).To ca ptur e a ppr oximate volumetric soil moistur e, we also collected 10 cm × 10 cm × 5 cm cubes into plastic bags.
Bulk soil was homogenized and sie v ed with a 2-mm sie v e and immediatel y anal yzed for av ailable forms of nitrogen (N-NO 3 − ,

Soil biogeochemistry and microbial quantity
Dissolv ed or ganic carbon (DOC) was extr acted with cold water (sample: water ratio of 1:5) by shaking at 20 • C for 1 h and subsequentl y anal yzed after tr ansport for total or ganic carbon on a LiquiTOC II (Elementar, German y).Water extr actable forms of inor ganic nitr ogen (N-NO 2 − , N-NO 3 − , and N-NH 4 + ; sample: water ratio 1:5) were determined immediately after sampling on the field station using photometric nitrite , nitrate , and ammonium test kits (Spectroquant Test, Merck, Darmstadt, Germany) and a field portable spectrophotometer (Hach Lange DR 2800, Germany).Their concentr ations wer e summed and r eferr ed to as mineral nitrogen (Nmin).Water extractable phosphorus (P-PO 4 3 − ) was determined as soluble r eactiv e phosphorus according to Murphy and Riley ( 1962 ) using a field portable spectr ophotometer (Hac h Lange DR 2800).Total content of elements in soil matrix (Si, Ca, Al, K, F e , Mg, P, and S) was determined by X-ray fluorescence (XRF) (Delta Premium XPD 6000, Olympus Innov-X, USA) under standard lab conditions (Tejnecký et al. 2015 ).Labile compounds (Ca The amount of 10 g of the 2-mm sie v ed sample was air-dried and treated with 30% hydrogen peroxide and the soil suspension w as allo w ed to react under heated conditions with subsequent additions of 30% hydrogen peroxide, until all soil organic matter was r emov ed.The soil suspension was then measured by laser diffraction method (LDM) using a Fritsch Analysette 22 MicroTrec plus in the range of 0.1-2000 μm (ISO 11277 2009 ).As the amount of clay particles was ov er estimated by the Fraunhofer theory, the pycnometer bottle method was added (Di Stefano et al. 2010 ).The fraction < 63 μm was oper ationall y defined as the sum of fine silt and clay, which is typically associated with the majority of the soil organic matter (Hemk eme yer et al. 2018 ) and considered to be to be important for soil stabilization through interactions between fine particles and organic carbon (Bird et al. 2002 ).
TOC and TN contents of the soil wer e measur ed using an elemental anal yzer (v ario MICRO cube, Elementar Anal ysensysteme GmbH).The detection limits were TOC (1 mg g −1 ) and TN (0.1 mg g −1 ) and it was not possible to determine TN content in samples younger than 54 years.Soil samples contained a significant amount of carbonates, which were removed prior to analysis via acid fumigation with HCl v a pours (Harris et al. 2001 ).Analyses of TOC and δ 13 C TOC contents of dried soil material were conducted with an NC Elemental anal yzer (ThermoQuest, Br emen, German y) connected to an isotope ratio mass spectrometer (IR-MS Delta X Plus, Finnigan, Bremen, Germany).
Carbon, nitrogen, and phosphorus in microbial biomass (C mic , N mic , and P mic ) were estimated using the c hlor oform-fumigation extr action method (Br ookes et al. 1982, 1985, Vance et al. 1987 ).The extraction of fumigated soil follo w ed the protocol for available nutrients described abo ve .Nutrient concentrations in microbial biomass were calculated as the difference between concentrations in fresh soil extract and fumigated soil extract.Results wer e corr ected for incomplete extr action of all C mic (K ec = 0.45), all N mic (Ken = 0.54), and all P mic (Kep = 0.4) (Brookes et al. 1982, 1985, Vance et al. 1987 ).
Tetraether lipids were extracted from six forefield soils (20 g w et w eight, one pooled sample for each forefield site) after the protocol of Hopmans et al. ( 2004 ).The analysis was conducted solely as a complementary measur e, without labor atory r epetition.Ther efor e, caution w as exer cised when e v aluating the data.Lipid extracts to assess the biomarkers were dried and stored fr ozen until anal ysis by liquid c hr omatogr a phy mass spectr ometry (Becker et al. 2015 ).Tetraether lipids were quantified relative to a C46 tetraether injection standard and the r elativ e r esponse of a caldarchaeol standard.The compounds measured include isopr enoidal gl ycer ol dibiphytan yl gl ycer ol tetr aethers having zero to three rings (iGDGT0-3; m/z 1302, 1300, and 1298), crenarchaeol (m/z 1292, biomarker for marine archea), and branched tetr aethers pr oduced by soil bacteria having varying degrees of rings and methylation (m/z 1018, 1020, 1022, 1032, 1034, 1036, 1046, 1048, and 1050).iGDGT having four rings (iGDGT4) was below the detection limit.The BIT index (br anc hed and isoprenoid tetr aethers index), whic h is a pr oxy for r elativ e contribution of terrestrial and marine organic matter input to sediments, was calculated as a ratio of the sum of all measur ed br anc hed tetr aethers v ersus cr enarc heol (Sc houten et al. 2013 ).

Soil enzymes anal ysis, DN A amplicon sequencing, and qPCR
Extracellular enzymatic activities were measured for five soil enzymes responsible for organic carbon, nitrogen, and phosphorus utilization with standard fluorometric technique (Marx et al. 2001 ) modified by Bárta et al. ( 2013 ).The activity of extracellular enzymes was determined in water extracts.Briefly, 0.5 g of soil was homogenized in 50 ml of distilled water via an ultrasonication.The soil suspensions (200 μl) were then tr ansferr ed to a 96-well micr oplate .T hen, 50 μl of substrates labeled with 4-methylumbelifferone (MUB) or 7-amino-4methylcoumarin (AMC) w ere added.Standar d curves w ere measured with MUB/AMC in soil slurries for e v ery sample separ atel y.Concentrations of MUB/AMC for standard curves were: 1, 5, and 10 μM.Micr oplates wer e incubated at r oom temper atur e for 2 h and fluorescence was measured every 30 min.All fluorescence measur ements wer e performed on the microplate reader INFI-NITE F200 (Tecan, Germany) using the excitation wavelength of 365 nm and emission wavelength of 450 nm.Enzyme activities wer e expr essed as nmol of liber ated MUF/AMC per hour per gr am of dry soil.The stoic hiometric anal ysis of enzymes was done according to Hill et al. ( 2012 ) and is based on the assumption that micr obes r elease extr acellular enzymes in order to gain needed C, N, and P nutrients from complex organic substrates .T he analysis compares ratios of C:N and N:P processing enzymes and the r esults may impl y whic h nutrients ar e mainl y limiting for the micr obial gr owth and pr osperity (Sc hmidt et al. 2016 ).Carbon pr ocessing enzymes (E C ) comprise β-glucosidase (GLU) and cellobiohydr olase (CELL) activity, nitr ogen pr ocessing enzymes (E N ) ar e the sum of leucin aminopeptidase (ALA) and chitinase (CHIT) activity and phosphorus (E P ) is r epr esented by phosphatase (PHO) acitivity.
The amount of 0.25 g was used for DNA extraction using Pow-erSoil DNA isolation kit (MO BIO Laboratories , USA).T he DN A w as quantified fluorimetrically using Qubit (Thermofisher Scientific, UK) following the standard protocol and kit for dsDNA quantification.The aliquots of DN A extracts w ere sent to SEQme lab (Prague, Czech Republic) for the pr epar ation of a library and sequencing using MiSeq platform.The Earth Microbiome Project (EMP) protocol was used for libr ary pr epar ation with modified universal primers 515FB/806RB (Ca por aso et al. 2011 ) and ITS1F/ITS2 (White et al. 1990 ) for prokaryotic 16S rRNA and fungal ITS1 amplicons, r espectiv el y.T he co v er a ge of pr okaryotic primer pair 515FB/806RB was additionally tested in silico using ARB Silva database release 128.The primer pair 515FB/806RB covers almost uniformly all major bacterial and archaeal phyla.Bacterial 16S rRNA raw pair-end r eads (150 bp) wer e joined using ea-utils to obtain r eads of ∼250 bp length.Quality filtering of reads was applied as previously described (Ca por aso et al. 2011 ).After quality filtering the sequences were trimmed to 250 bp.We obtained 720 625 bacterial and 1746 017 fungal sequences after quality trimming and filtering.Before pic king the oper ational taxonomic units (OTU), the fungal ITS1 region was extracted from reads using ITSx algorithm (Bengtsson-Palme et al. 2013 ).Both 16S and ITS1 amplicons were trimmed to equal lengths in order to avoid spurious OTU clusters (Edgar 2013 ).Taxonomy was assigned to each read by accepting Silva119 taxonomy string of the best matching Silva119 sequence.Fungal reads wer e cluster ed to OTUs using open-r efer ence OTU pic king pr otocol (sequence similarity 98.5%) using UNITE ver.8.0 database (Koljalg et al. 2013 ).Blast algorithm (e-value ≤ 0.001) was used for taxonomic assignment.FAPROTAX (Louca et al. 2016 ) and FUNguild (Nguyen et al. 2016 ) algorithm was then used for bacterial functional annotation and the fungal lifestyle assignments, respectiv el y.Raw sequences were deposited at ENA under the project n.PRJEB51542.
Quantification of bacterial and fungal SSU rRNA genes was performed using the FastStart SybrGREEN Roche ® Supermix and Step One system (Life Tec hnologies, USA).Eac h r eaction mixtur e (20 μl) contained 2 μl DNA template ( ∼ 1-2 ng DNA), 1 μl each primer (0.5 pmol μl −1 each, final concentration), 6 μl dH2O, 10 μl FastStart SybrGREEN Roche ® Supermix (Roche, France), and 1 μl BSA (F ermentas , 20 mg μl −1 ).Initial denaturation (3 min, 95 • C) was follo w ed b y 30 c ycles of 30 s at 95 • C, 30 s at 62 • C, 15 s at 72 • C, and completed by fluorescence data acquisition at 80 • C used for target quantification.Product specificity was confirmed by melting point analysis (52-95 • C with a plate read every 0.5 • C) and amplicon size was verified with agarose gel electrophoresis.Bacterial standards consisted of a dilution series (ranging from 10 1 to 10 9 gene copies μl −1 ) of a known amount of purified plasmid where PCR product using the SSU gene-specific primers 341F/534R (Muyzer et al. 1993 ) was inserted.R 2 values for the standard curves were > 0.99.Slope v alues wer e −> 3.37 giving an estimated amplification efficiency of > 93%.The qPCR conditions for fungal quantification were as follo ws: initial denaturation (10 min, 95 • C) follo w ed b y 40 c ycles of 1 min at 95 • C, 1 min at 56 • C, 1 min at 72 • C, and completed by fluorescence data acquisition at 72 • C used for target quantification.Fungal DN A standar ds consisted of a dilution series (ranging from 10 1 to 10 7 gene copies μl −1 ) of a known amount of purified plasmid where PCR product using the SSU gene-specific primers n u-SSU-0817-5 and n u-SSU1196-3 (Borneman and Hartin 2000 ) was inserted.R 2 values for the fungal standard curves were > 0.99.The slope was between −3.34 and −3.53 giving estimated amplification efficiency between 95% and 93%, r espectiv el y.Detection limits for the assays (i.e.lo w est standar d concentration that is significantl y differ ent fr om the nontemplate contr ols) wer e less than 100 gene copies for each of the genes per assa y.Samples , standards and nontemplate controls were run in duplicates.To deal with potential inhibition during PCR the enhancers BSA and DMSO were added to the PCR mixtur e.Also, se v er al dilutions (10x, 20x, 50x, 100x, and 1000x) for each sample were tested to see the dilution effect on Ct values.Alpha diversity metrics the Chao1 ric hness (measur ement of OTUs expected in samples given all the bacterial/fungal species that were identified in the samples), were calculated after rarefying all samples to the same sequencing depth of 3200 and 2800 sequences for bacterial and fungal comm unity, r espectiv el y (Chao 1984 ).

Sta tistical anal yses
The effect of the northern versus southern forefield on soil parameters and on par ameters c hanges in time were tested using a generalized linear mixed-effect model with Gamma distribution and logarithmic link function and a likelihood-ratio test, which was run in R version 4.0.2(R Core Team 2020 ) in R pac ka ge stats .The zone identity for eac h for efield was used as a random effect (i.e.eight groups).The significant differences in time were evaluated separ atel y for eac h for efield using Tukey's HSD post hoc tests in the R pac ka ge multcomp to examine pairwise differences between the zones (different soil a ges).Spearman corr elation coefficients were determined in R package ggpubr to assess how str ongl y c hosen par ameters wer e r elated to eac h other.The effect of for efield on the total content of chosen soil elements determined by XRF and on labile compounds was visualized using principle component analyses in CANOCO for Windows 5.0 (Ter Braak and Šmilauer 2012 ) with zone (age) as a co variate .In all analyses from CANOCO, only the adjusted explained variation is presented in the text.In CANONO, variation partitioning was also applied to quantify the unique effects of TOC, bedr oc k (total content of elements in soil matrix determined by XRF-Si, Ca, Al, K, F e , Mg, P, and S) and available nutrients (DOC, P-PO 4 3 − , Nmin, Ca 2 + , Mg 2 + , and K + ) on the microbial quantity (C mic , bacteria, and fungi) and quality (CNP pr ocessing enzymes), separ atel y during 79 and 10 000 years of soil succession.Subsequently, a forw ar d selection pr ocedur e was also performed to identify the single parameters that best explain the microbial quantity and quality and only P -values adjusted with Holms correction were considered.Both analyses were run with zone (age) as co variate .To estimate the beta-diversity in soil micr obial comm unities and in their functional anal yses, nonmetric multidimensional scaling (NMDS) ordinations wer e gener ated using CANOCO on the basis of Bray-Curtis dissimilarities of squarer oot tr ansformed r elativ e abundances of O TUs .

Soil properties
XRF analysis of main soil elements has identified Si to be a prevailing element in the soil matrix of both forefields (from 13.6% to 22.5%), follo w ed b y calcium (from 3.5% to 8.3%) and/or aluminium (from 2.6% to 6.0%) ( Table S1 , Supporting Information ).The significant difference in the content of most soil elements between northern and southern forefield corresponds to the overall lithological distinction described in detail within the methods (Fig. 2 A; Table S1 , Supporting Information ).This geochemical distinction is further evident in the higher amount of water extractable cations and in higher content of fine soil particles < 63 μm of the more easily weatherable sedimentary rocks, which ar e pr esent in the glacial till of the southern forefield (Fig. 2 B; Table S2 , Supporting Information ).The higher proportion of fine particles < 63 μm in the soil matrix correlates with a higher soil moisture observed in the whole southern forefield ( r = 0.7, P < .05;Table S2 , Supporting Information ).Besides the geochemical distinction, forefields did not significantly differ in pH, TOC, TN, or water extractable C and P, but they differed in water extractable N across all sites ( Table S2 , Supporting Information ).All samples were alkaline with pH between 8.28 and 9.81.TOC le v els sho w ed very distinct trends in time within each forefield.TOC gradually increased with age on the northern forefield, from low values at the N1 site (0.11%) to high values at the NR site (2.78%) ( Table S2 , Supporting Information ).In contrast, on the southern forefield, TOC was higher at the S1 site (0.16%) than at the N1 site (0.11%), decreased at the S2 site (0.09%), and gr e w in the later stages (S3 0.19%, SR 3.25%) (Fig. 3 A; Table S2 , Supporting Information ).TN was below the detection limit in sites younger than 54 years and then increased on both forefields ( Table S2 , Supporting Information ).All water extractable nutrients generally increased with time, only SO 4 2 − had the opposite trend.Both forefields contained a proportion of sulphate miner als, whic h r eleased especiall y high amounts of SO 4 2 − on the S1 site together with water extractable Ca, Mg, and K ( Table S2 , Supporting Information ).
The stable C isotope composition of TOC ( δ 13 C TOC ) varied from −20.8 ‰ to −24.3 ‰ ( Table S3 , Supporting Information ).TOC samples collected on the southern forefield were typically more depleted in 13 C than on the northern forefield, and N1 site exhibited the highest δ 13 C TOC values ( −20.8 ‰).The interpretation of the biomarker data is constrained by the limited replication, as only one pooled sample was used for each forefield site.Howe v er, despite this limitation, we have included the data in order to propose a broader range of explanations for the various successional de v elopments observ ed.The BIT index on the northern forefield steadily increased with soil age (0.39-0.59;Table S3 , Supporting Information ), suggesting increasing inputs of terrestrial (brGDGT) r elativ e to pela gic/glacial (cr enarc haeol) OM ov er F igure 2. RDA or dinations of geoc hemical par ameters of inv estigated soils constr ained by the attribution to the for efield: (A) total content of main soil elements determined by XRF analysis (pseudo-F = 45.3,P < .01),(B) Water extractable nutrients and fine soil particles < 63 μm (pseudo-F = 15.7,P < .01).Soil age (Zone) was used as a covariate for both analyses.time (Hopmans et al. 2004 , Brady andDaniel 2013 ).In contrast, the BIT index was consistently high on the southern forefield ( > 0.69; Table S3 , Supporting Information ) suggesting r elativ el y higher inputs of OM from terrestrial sources .T he organic matter on the southern forefield generally comprised higher content of monoand disaccharides, fatty acids, and sitosterol and brassicasterol ( Table S3 , Supporting Information ).

Microbial quantity and stoichiometry
Distinct tempor al tr ends in TOC dynamics within eac h for efield wer e r eflected in (i) micr obial abundance expr essed as C mic , (ii) the quantity of bacteria and fungi expressed as SSU gene copies per gram of dry soil, and (iii) bacteria species richness (Fig. 3 ).These micr obial par ameters differ ed the most betw een the y oungest sites (i.e.N1 and S1).Values were the lowest at N1 sites, whereas southern S1 sites were characterized by significantly higher microbial biomass and bacterial and fungal abundance (Fig. 3 ).A significant drop of microbial biomass was observed at southern S2 sites, thus the drop in microbial quantity was significant only for fungi, not for bacteria (Fig. 3 ).The trends and values in the older sites ( > 54 years) were similar in both forefields (Fig. 3 B-D).Bacterial species richness was equivalent or increased with time on both forefields (Chao index: northern forefield from 890 to 1396, r espectiv el y, southern for efield fr om 1306 to 1648, r espectiv el y) (Fig. 3 E), and was consistently higher in the southern forefield.Ho w e v er, fungal species richness in the youngest plots showed a completely different trend between the two forefields (Fig. 3 F).In the southern foreland, the Chao index was significantly lo w er in the youngest plots S1 (679) and S2 (616) compared to the oldest plots S3 (480) and SR (513).The fungi in the northern foreland sho w ed a completely opposite trend (Fig. 3 F).
Forefields also differed in C mic normalized to TOC, with extr emel y low v alues at the N1 sites (9.6 ± 11.0), compared to the significantl y higher v alues at the S1 sites (143 ± 64.8) (Fig. 4 A).Ho w e v er, similarl y to TOC, most of all micr obial par ameters conv er ged at N3 and S3 sites (after 79 years) and the forefields dev elop similarl y in later sta ges.Fungi to bacteria r atio was v ery low in the youngest sites of both forefields (N1, N2, S1, and S2; from 7.24 × 10 −5 to 4.00 × 10 −4 ), indicating the general prevalence of bacterial over fungal communities.In later stages, especially in S3 sites (1.54 × 10 −3 ), an expansion of fungi was observed (Figs 3 D and 4 B).Similarly to C mic , also N mic and P mic were consider abl y higher at the S1 site (21.7 μgN g dw −1 and 5.5 μgP g dw −1 , r espectiv el y) compar ed to the N1 site (0.95 μgN g dw −1 and 1.5 μgP g d w −1 , respecti vely) and did not change significantly at the S2 and S3 sites except of a decrease in N mic at S2 sites, while they graduall y incr eased on the northern for efield ov er time (Fig. 4 C and D; Figure S1D , Supporting Information ).

Enzyme activity and stoichiometry
The sum of enzyme activity increased along the northern forefield, while there was no clear pattern on the southern forefield (Table 1 ).Despite remarkable differences in the sum of enzyme activity per gram of soil among N1 (31 nmol h −1 g −1 ) and S1 sites (359 nmol h −1 g −1 ), the v alues wer e similar when normalized to microbial biomass (2.82 nmol h −1 g −1 and 2.86 nmol h −1 g −1 , r espectiv el y).At these youngest sites, phosphatase (PHO) was the main processing enzyme measured (84.3% and 59.5%, respectiv el y), but its pr oportion decr eased in time on behalf of N processing enzymes (Table 1 ).Carbon acquiring enzymes (GLU and CELL) had very low activities across both c hr onosequences until the r efer ence sites.Stoic hiometric anal ysis of CNP pr ocessing enzymes (Hill et al. 2012 ) r e v ealed a clear distinction of the youngest deglaciated N1 and S1 sites in terms of potential nutrient limitation (Fig. 5 ).While the northern forefield appeared to be C and P colimited, the southern forefield was mainly P limited.Later, both forefields sho w ed a similar tr end of gr adual shift fr om P limitation (S1 and N2 sites) to N limitation (S2, S3, and N3 sites) and finally to almost no nutrient limitation (S3, SR, and NR sites).

Effect of chemical parameters on microbial quantity and enzyme activity
Both microbial quantity (microbial biomass, abundance of bacteria and fungi determined by qPCR) and enzyme activities were str ongl y positiv el y corr elated with soil a ge (C mic r = 0.72, bacteria r = 0.68, fungi r = 0.82, and enzymes r = 0.66, all P < .001)and with TOC (C mic r = 0.86, bacteria r = 0.75, fungi r = 0.81, and enzymes r = 0.63 r espectiv el y, P < .001).The exception was the soil de v elopment within 79 years in the S1-S3 sites, where the correlations of microbial parameters with age were not significant and the soil de v elopment was clearl y driv en by differ ent par ameters.Variation partitioning anal yses of both for efields r evealed that, together with time and TOC, bedr oc k type and available nutrients also played an important r ole.Specificall y, bedr oc k (16.5%) and available nutrients (10.5%) explained together more  variability than TOC (18.9%) during the first 79 years of soil succession (Fig. 6 ).
The forw ar d selection of single environmental parameters revealed TOC to be the only explanatory variable of the microbial quantity (47.7%, pseudo-F = 58.3,P = .022)and microbial activity (53.6%, pseudo-F = 73.8,P = .022)across all sites.Interestingly, onl y micr obial activity of the southern for efield was significantl y influenced also by P-PO 4 3 − (7.4%, pseudo-F = 6.4,P = .03).This available phosphorus seemed to be especially important for the abundance of fungi, which sho w ed a close correlation (r = 0.72, P < .001).

Microbial community composition and functioning
After quality trimming and filtering, we obtained 737 010 (7046 OTUs) and 1521 308 (2774 OTUs) high quality sequences for bacterial and fungal comm unities, r espectiv el y.We wer e able to taxonomically affiliate 3932 bacterial OTUs (56%) and 602 fungal OTUs (22%) up to the genus le v el.In the fungal community, we found 535 OTUs (19%) that wer e taxonomicall y assigned onl y to the domain le v el.These fungal species might r epr esent nov el, unkno wn ar ctic species that are still not r epr esented in r ecent fungal databases.Bacteria and fungi were rarefied to 3212 and 2866 sequences per sample, r espectiv el y.NMDS analysis sho w ed that the bacterial communities w ere different between the early and late stages (10 kyr) and were also different between the two forefields (Fig. 7 A).In contrast, functional diversity was only different between the early stages and the late (10 kyr) stage but was not as markedl y differ ent between the forefields .T hus , our data sho w ed that (i) although the bacterial communities of the early-stage forefields were distinct, they exhibited a higher degree of functional overlap and (ii) that the two forefields became more similar in both species composition Table 1.Enzyme activities.and function after about 10 kyr.This trend of community convergence was consistent with the data from chemical analyses of the soils of the two forefields ( Table S2 , Supporting Information ).

Site
Functional analyses further sho w ed critical differences in trends of specific fungal lifestyles, mainly the proportions of lichenized fungi between the northern and southern forefield ( Figure S2 , Supporting Information ).Lichenized fungi in the youngest southern S1 sites r epr esented 55% of the community, on av er a ge, and steadil y decr eased with time.In contrast, they represented only 12% in the youngest northern N1 sites and gradually incr eased fr om N1 to N3 sites.
The high proportion of OTU of lichenized fungi at the youngest southern S1 sites was accompanied by r elativ el y higher pr oportions of c y anobacteria and diazotrophs ( Figure S1 , Supporting Information ).These functional guilds decreased with time on the southern forefield while an opposite trend was seen on the northern forefield.This can relate to decreasing trends of C and N flux to the soil in the southern for efield, pr esumabl y via lo w er CO 2 and N 2 fixation performed by cyanobacteria and diazotrophs.

Discussion
Global warming has accelerated the retreat of glaciers in polar ecosystems and the exposure of new terrain that may be subject to succession by many species, including microbial species.Studies in Alaska have shown that limited nutrient availability on poor geological substrates after the glacier retreat may be more important for the early microbial succession than climatic conditions, suc h as temper atur e and pr ecipitation (Sc hmidt et al. 2016 ).T hese studies ha ve also highlighted that the influence of local climate is difficult to separate from that of nutrient availability (Nemergut et al. 2005 ).Both questions r emain unanswer ed because the available data were mostly compared between distant glacier for efields, wher e the influence of climatic conditions could not be a ppr opriatel y filter ed out (Lazzar o et al. 2009, Bajerski and Wa gner 2013, Alfar o et al. 2017 ).
Our study pro vides , for the first time, a detailed insight into the biogeochemical and microbial succession in two closely spaced forefields of the retreating Nordenskiöldbreen (Svalbar d, Norw ay) with similar climatic conditions but different bedrock geology (Fig. 2 A).Given the close proximity of the two forefields, this study offers a unique possibility to test the hypothesis that the substrate quality is critically important for microbial succession during the first decades after deglaciation (Schmidt et al. 2016 ) and may consider abl y influence the de v elopment of micr obial comm unities and biogeochemistry in later successional stages of soil evolution.

Effect of mineral substrate on nutrient availability
Our study confirmed that differ ent substr ate par ameters and nutrient availability resulted in different soil succession of the micr obial comm unity in nearby for efields .T he most pronounced differ ences wer e in miner al substr ate composition (Fig. 2 ).While the mineral substrate of the northern forefield was dominated by glacially formed rocks and thin glacial deposits with silicate r oc ks, the southern forefield was completely covered by thick glacial deposits with high proportion of carbonates.Carbonate sediments typically weather more rapidly and can increase the content of fine soil particles < 63 μm, leading to higher soil moisture ( Table S2 , Supporting Information ) and higher nutrient availability for microbes (Fig. 2 B).The higher content of simple saccharides and available fatty acids ( Table S3 , Supporting Information ) and higher content of water-extractable Ca, Mg, and K (Fig. 2 B; Table S2 , Supporting Information ) in the southern forefield indicated higher quality OM that can be more rapidly utilized by micr oor ganisms, leading to higher microbial biomass in the southern for efield, especiall y at the youngest sites.Our data, ther efor e, show that substrate chemistry and nutrient availability are more important for microbial activity and early soil de v elopment (Figs 2  and 6 ) than the commonly reported TOC (Fig. 7 A) (Tytgat et al. 2016, Kim et al. 2017 ).
Ho w e v er, the abov e-mentioned differ ences between the for efields do not provide us with any information on whether C, N, or P ar e av ailable in sufficient quantities for micr oor ganisms and whether they are limiting for their growth and development.To answer this question, we used an enzymatic model that provides information on the utilization of C, N, and P in the context of their limitation for micr oor ganisms and their enzymatic activity (Fig. 5 ) (Hill et al. 2012 ).Enzymes of heter otr ophic bacteria and fungi involved in C, N, and P processing sho w ed distinct trends, especially in early successional stages on both forefields.We found a shift from P to N limitation in the southern for efield, wher eas the northern forefield was initially more colimited by C-P and shifted to greater N limitation in later sta ges, whic h is consistent with a pr e vious study (Bueno de Mesquita et al. 2020 ).Ther efor e, we hypothesize that the lo w er complexity and higher quality of Cric h or ganic material on the southern for efield r equir ed less investment by microorganisms in C enzymes to obtain energy and carbon, which allo w ed microbes to grow faster in the early stages but resulted in limitation of N and P.This can also be observed in differ ent micr obial biomass and abundance of bacteria and fungi in these forefields .T he later shift to N limitation on both forefields corr obor ates other observ ations and a gener al par adigm of higher N r equir ements with mor e de v eloped soils , among others , in association with increasing vegetation cover (Vitousek et al. 1993, Bueno de Mesquita et al. 2020 ).The different character of the organic material was further supported by an increasing BIT index and decreasing δ 13 C with age in the northern forefield, indicating gr adual accum ulation of soil-deriv ed or ganic material, as could be expected along a classical c hr onosequence .T hese findings are consistent with reports of taxa mainly from supra-and subglacial habitats in front of the Damma Glacier, Switzerland (Rime et al. 2016a ) or a gradual transition from glacial to edaphic taxa in front of the Fourcade Glacier, maritime Antarctica (Gyeong et al. 2021 ).We ar e awar e that the inclusion of biomarker data with low replication (one pooled sample for each forefield site) needs to be justified.Based on our results, we would like to suggest that biomarker distributions may help to unr av el micr obial assembly dynamics that are still poorly understood and may not be full y ca ptur ed by measuring soil physico-c hemical par ameters alone.

Different succession of the microbiome in the northern and southern forefield
In Arctic extr eme envir onments, the successional pattern deviates from the classical model of directional change and species r eplacement (Matthe ws 1978 , Svoboda and Henry 1987 ).Under excessiv e climatic str ess, competition is r educed and dir ectional, nonsubstituting succession is mor e common, with nativ e species remaining and new species being added during succession (Svoboda andHenry 1987 , Jones andHenry 2003 ).Pr e vious studies fr om Sv albard glacier for efields suggest that colonization of both plants and root-bound fungi follow this model of directional, nonsubstituting succession (Hodkinson et al. 2003, Davey et al. 2015 ).
Our data show that the bacterial and fungal microbiomes of the northern and southern for efields e volv ed differ entl y in biomass, composition, and activity during the early stages of deglaciation.This different development lasted roughly until the first 79 years after deglaciation.T hereafter, con vergence occurred and the differences between the forefields almost disappeared (Figs 3 -5 and 7 ).Total microbial biomass was significantly higher on the youngest S1 sites than on the N1 sites (Fig. 3 B).Compared to the northern, the southern microbiome experienced a significant decline in both abundance and diversity over a relatively short period of time, pr obabl y r elated to the decline of fungi (Fig. 3 D) and pr obabl y also related to a significant rebuilding of the fungal microbiome (Fig. 3 F; Figure S2 , Supporting Information ).The Chao index sho w ed very lo w fungal richness of the y oungest southern sites (Fig. 3 F), indicating a dominance of few fungal species .T hus , the later increase in species richness may be a result of community r earr angement.Fungal comm unities de v eloped differ entl y from the beginning on both forefields, and differences were also reflected in the relative abundance of specific functional groups of fung i. Lichen-forming fung i are often found in early successional stages after glacial retreat (Syers and Iskandar 1973, Zumsteg et al. 2012, Garrido-Benavent et al. 2020 ), and they also str ongl y and r elativ el y r a pidl y dominated the micr obiome of the southern for efield, whic h corr elates with higher abundance of bacterial phototr ophs ( Figur e S1 , Supporting Information ) and with higher le v els of sitoster ol and br assicaster ol ( Table S3 , Supporting Information ), biomarkers indicating algal and/or plant phytomass (Volkman 2003, Rontani et al. 2012 ).Ho w e v er, the lic hen-forming fungi abundance in the fungal community declined significantly over time on southern forefield and they were gradually replaced by sa pr otr ophs and partiall y ericoid species in the oldest sites after deglaciation (Selosse et al. 2018 ).T hus , the fungal microbiome of the southern forefield experienced mor e pr onounced fluctuations in both abundance and diversity, and a significant rebuilding of the fungal microbiome was observed, indicating a direct evolution with partial replacement of fungal species compared to the northern forefield with a rather nonsubstituting succession, more common for the High Arctic (Svoboda andHenry 1987 , Jones andHenry 2003 ).
In contrast to the fungal microbiome, bacterial abundance and ric hness incr eased gr aduall y with soil de v elopment on both for efields, consistent with other studies (Schütte et al. 2010, Jiang et al. 2018, Gyeong et al. 2021 ).Howe v er, we found specific differences in some major functional groups that might be important for initial succession in soil.The microbiome of the northern forefield had a higher proportion of bacteria degrading more complex and less available compounds in the early stages of degradation.This was mainl y r eflected by a higher proportion of cellulolytic and c hitinol ytic bacteria ( Figur e S1 , Supporting Information ) and pr obabl y r elated to lo w er nutrient availability and higher C mining (Fig. 5 ; Table S2 , Supporting Information ).In contrast, the young southern forefield contained a higher proportion of phototrophic and nitrogen-fixing bacteria ( Figure S1 , Supporting Information ), also supported by lower δ 13 C TOC values ( Table S3 , Supporting Information ).These two k e y functional guilds in the southern forefield mediate the flux of atmospheric C and N during early soil succession to microbial community.When they have sufficient P for energy production (Dar c y and Schmidt 2016 , Dar c y et al. 2018 ), they 'pump' available C and N into the system, allowing other heter otr ophic micr obes to de v elop (Cha pin et al. 1994 ).Indeed, more P mic was measured on the southern forefield (Fig. 4 D), suggesting that higher availability of P promoted the de v elopment of heter otr ophic bacteria and fungi and increased the overall microbial biomass in the earl y sta ges of deglaciation compared to the northern forefield.Our findings are consistent with other studies that have found that P is essential for accelerating phototr ophic gr o wth (Dar c y and Schmidt 2016 , Bueno de Mesquita et al. 2020 ) and for microbial succession in general (Knelman et al. 2014 ).T hus , the two forefields evolved quite differently in terms of the number, composition, and functional potential of the fungal and bacterial microbiome in the early stages up to 79 years after glacial r etr eat.

Different trends in convergence and redundancy of bacteria and fungi over time
Similarly to other studies (Rime et al. 2016b, Alfaro et al. 2017, Jiang et al. 2018, Garrido-Benavent et al. 2020, Vimercati et al. 2022 ), the trajectories of bacteria and fungi sho w ed different patterns during succession (Fig. 7 ).As discussed abo ve , the bacterial and fungal microbiomes were clearly affected by the geochemistry and nutrient availability in the youngest sites (Fig. 7 ).Only bacteria sho w ed a gr adual conv er gence with time, wher eas the lack of a clear pattern of fungal assembly is consistent with previous studies (Brown andJumpponen 2014 , Castle et al. 2016 ).Fungal succession is thought to be controlled by stochastic processes (Jiang et al. 2018, Gyeong et al. 2021, Lin et al. 2022, Vimercati et al. 2022 ), which may be caused by their different dispersal possibilities or their close relationships with vegetation (Schmidt et al. 2014 ), together with high geomorphological and geological heterogeneity of the glacial forefields (Ozerskaya et al. 2009, Wojcik et al. 2020, Gyeong et al. 2021 ).Compared to the microbial composition, functional groups of bacteria and fungi were shaped much less by the geochemistry and exhibited partial functional redundancy.
In summary, in the northern forefield, most geochemical and micr obial par ameters gr aduall y incr eased with time and may be considered as the classical chronosequence reported by most pr e vious studies (Cr oc ker and Major 1955, Cha pin et al. 1994, Bernasconi et al. 2011 ).In contrast, in the southern forefield, nutrients and microbial biomass were already high at the youngest sites, leading to significant later fluctuations in bacterial and fungal abundance and diversity that last for a ppr oximatel y the first 79 years after deglaciation.
We suggest that after deglaciation in the southern forefield, the earl y micr obial comm unities thriv ed on the higher availability of n utrients, most lik el y pr ovided by the miner al substr ate and microbial guilds involved in CO 2 and N 2 fixation (mainly lichenized fungi and oxygenic phototrophs) and, therefore, evolved much faster.Subsequently, n utrient de pletion occurred rapidly, dri ving the habitat to nutrient limitation, and the microbial biomass again declined and rearranged in composition and in functional potential (e.g.lichen-forming fungi were replaced by saprotrophic fungi) as partially observed in another study (Schmidt et al. 2016 ).Our findings support the hypothesis that differ ent bedr oc k may acceler ate earl y soil succession after deglaciation, providing limiting nutrients during micr oor ganism assembl y, ther eby pr omoting more rapid soil stabilization and providing higher quality organic matter.Our study also sho w ed distinct patterns of bacterial and fungal trajectories during succession.The chemical parameters and microbiomes of both forefields converged gradually with time, while the fungi did not show a clear pattern.

Figure 1 .
Figure 1.Northern and southern forefield of the Nordenskiöldbreen with black triangles as sampling sites and marked zones of deglaciation based on the satellite imagery provided by © Norsk Polar Institute ( http://www.npolar.no ) and map background from TopoSvalbard ( http://toposvalbar d.npolar.no).The Nor denskiöldbreen glacier front is from summer 2009 and N1 sites were already deglaciated during the sampling campaign.

Figure 3 .
Figure 3.Effect of forefield and time on the microbial quantity determined by generalized linear mixed-effect model with Gamma distribution: (A) total organic matter, (B) carbon in microbial biomass, (C) amount of bacteria, (D) amount of fungi, (E) Chao index of bacteria, and (F) Chao index of fungi.Asterisks next to the title indicate significant difference between the forefields ( * ∼ P = .01-.05, NS ∼ not significant).Different letters indicate significant differences among sites of different age within each forefield ( P < .05,multiple comparisons using Tuk e y's post hoc test).Boxplots visualize summary statistics (the median and the first and third quartiles) with whiskers and outlying points.Northern (red) and southern (blue) sites are described together with samples age: N1 and S1 = 0-25, N2 and S2 = 26-54, N3 and S3 = 55-79, and NR and SR = 10 000.

Figure 4 .
Figure 4. Effect of forefield and time on the microbial quantity determined by generalized linear mixed-effect model with Gamma distribution: (A) C biomass to TOC, (B) fungi to bacteria ratio, (C) nitrogen in microbial biomass, (D) phosphorus in microbial biomass.Asterisks next to the title indicate significant difference between the forefields ( * ∼P = .01-.05, NS ∼ not significant).Different letters indicate significant differences among sites of differ ent a ge within eac h for efield ( P < .05,m ultiple comparisons using Tuk e y's post hoc test).The bo xplots visualize summary statistics (the median and the first and third quartiles) with whiskers and outlying points.Northern (red) and southern (blue) sites are described together with samples age: N1 and S1 = 0-25, N2 and S2 = 26-54, N3 and S3 = 55-79, and NR and SR = 10 000.
of a forefield on enzyme activities ( * * * ∼ P < .001,* * ∼ P < .01,* ∼ P = .01-.05, NS ∼ not significant) determined with a glmer model with a Gamma distribution and a logarithmic link function and a likelihood-ratio test.Effect of time on enzyme activities was e v aluated separ atel y for eac h for efield using Tuk e y's HSD post hoc tests (different letters indicate significant differences; P < .05 between sites within the forefield).Brackets indicate ± standard error (within each site, n = 9), n.d.∼ not detected.Inter pr etation: carbon enzymes = sum of β-glucosidase (GLU) and cellobiohydrolase (CELL) activity, phosphorus enzymes = phosphatase (PHO) acitivity, and nitrogen enzymes = sum of leucin aminopeptidase (ALA) and chitinase (CHIT) activity.

Figure 6 .
Figure 6.Variation partitioning analyses-the unique explained variability of TOC, bedrock, and available nutrients in (A) microbial quantity and (B) micr obial activity.Anal yses wer e made separ atel y for 10 000 y ears and 79-y ears-old soil succession.TOC = total organic carbon, Bedr oc k = main soil elements measured with XRF (Al, F e , Si, Mn, P, S, Mg, and Ca), and water extractable nutrients = DOC, PO 4 3 − , Nmin, Ca 2 + , Mg 2 + , and K + .In all analyses age of the soil was used as co variate .
• N, 16.78 • E) is a polythermal valley glacier and largest outlet glacier of Lomonosovfonna Ice Cap in Billefjorden area